Quantitation of individual protein kinase activity

a technology quantitation, applied in the field of individual protein kinase activity, can solve the problems of extended assay completion time, inability to produce similar antibodies to phosphoserine and phosphothreonine, and limited detection of tyrosine kinases

Inactive Publication Date: 2000-05-23
PROMEGA
View PDF2 Cites 50 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A disadvantage of the dot blot method is that it is limited to detecting tyrosine kinases.
Attempts to produce similar antibodies to phosphoserine and phosphothreonine have not been successfully employed to assay for phosphoserine- and phosphothreonine-containing proteins.
In addition, the assay requires several incubation and washing steps, each taking a considerable amount of time, which results in a greatly extended assay completion time.
The colored dot may limit the ...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Quantitation of individual protein kinase activity
  • Quantitation of individual protein kinase activity
  • Quantitation of individual protein kinase activity

Examples

Experimental program
Comparison scheme
Effect test

example 1

Kinase Activity of cAMP-Dependent Protein Kinase

The kinase activity of cAMP-dependent protein kinase (PKA) was carried out in duplicates at each of the various peptide substrate concentrations, in a reaction volume (50 .mu.L) containing 40 mM Tris HCl, pH 7.4, 20 mM MgCl.sub.2, 100 .mu.M .gamma.-.sup.32 P-ATP (sp.act. of 100-200 cpm / pmol), 100 .mu.g / ml of bovine serum albumin (BSA), and the appropriate biotinylated peptide substrate: *-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Promega Peptide A, SEQ. ID. NO: 1, M.Wt of 998.4).

The reaction was started by addition of the appropriate amount of enzyme (Promega #V5221) to give 2 Kemptide units and incubated for 0, 5, and 10 minutes at 37.degree. C. (one Kemptide unit is defined as the amount of enzyme required to catalyze the incorporation of 1 pmol of phosphate into Kemptide substrate in 1 minute). At specified periods of time at the appropriate temperature, the reaction was terminated with the addition of 10 .mu.L of terminating solution as describ...

example 2

Kinase Activity of PKA in Rat Tissues

The kinase activity of PKA in various rat tissues was also investigated using the modified peptide substrate and assay protocol as described above.

As illustrated in FIG. 5, the protein kinase activity of PKA was 2.2, 1.75, 3.2, and 1.9 nmol .sup.32 P / min / mg of rat liver, ovary, heart, and brain, respectively, and was comparable to those obtained with unmodified peptide and the phosphocellulose paper method. The phosphate incorporation into the modified substrate was completely abolished when 100 .mu.M of PKA inhibitor (PKI) was included in the reaction. Thus, the phosphorylation of the modified peptide substrate, under the conditions described above and assayed with the protocol, is catalyzed by PKA and not by other protein kinases present in the tissue extract.

Any protein(s) that is phosphorylated in the reaction and has a basic charge will not bind to the matrix. Only the phosphorylated modified peptide substrate is bound to the matrix. Thus, t...

example 3

Protein Kinase Activity of Ca.sup.2+ and Phospholipid Dependent Protein Kinase

The protein kinase activity of Ca.sup.2+ and phospholipid dependent protein kinase activity (PKC) was determined in a similar manner to that of PKA described above, except that the biotinylated peptide substrate was a derivative of neurogranin; namely, biotinylated neurogranin .sub.(28-43) Chen, S-J, et al., 1993.

The peptide was modified by the addition of biotin containing moiety: *-Ala-Ala-Lys-Ile-Gln-Ala-Ser-Phe-Arg-Gly-His-Met-Ala-Arg-Lys-Lys (Promega Peptide B, SEQ. ID. NO: 2, Mol.Wt. is 2139.6), and was used at a concentration of 100 .mu.M. The enzyme (#V5261, Promega Corporation) was diluted 5.times. in 100 .mu.g / ml of BSA.

The kinase activity of the PKA was determined in a reaction volume of 50 .mu.L consisting of 40 mM Tris. HCl, pH 7.4, 10 mM MgCl.sub.2, BSA at 100 .mu.g / ml, and 100 .mu.M of .gamma.-.sup.32 P-ATP (sp.act. of 100 cpm / pmol). The activity was determined in the presence and absence of...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Lengthaaaaaaaaaa
Timeaaaaaaaaaa
Radioactivityaaaaaaaaaa
Login to view more

Abstract

A method of quantitating the activity of a selected protein kinase on a peptide substrate is provided. The peptide substrate is conjugated to a binding compound. The modified peptide substrate is then added to a solution containing the selected protein kinase. The protein kinase and the peptide are incubated along with a label for sufficient time to form a modified peptide product having the binding compound and the label. The modified peptide product is then bound to a matrix having a high binding compound affinity. The bound peptide is then washed and the activity of the protein kinase is measured. Also provided is a kit for the stated method.

Description

The invention is directed to a process for providing an assay protocol that measures enzymatic activity. More particularly, the invention is directed to a process to precisely and conveniently quantitate enzymatic activity of protein kinases and further to provide an assay specific for individual protein kinases in the presence of other protein. kinases.CITED REFERENCESA bibliography of the references cited in this application can be found in the section preceding the claims.DESCRIPTION OF THE PRIOR ARTEnzymes are large proteins that catalyze reactions in living cells. Enzymes build up or tear down other molecules. For example, enzymes catalyze the synthesis of fat from fatty acids, form complex sugars from glucose and fructose, and aid in the formation of other proteins from amino acids. Enzymes also reverse the build-up process by breaking down more complex structures. Enzymes are generally specific to certain substrates for their reactions. For example, an individual enzyme may c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K7/00C07K7/08C07K7/06C12Q1/48G01N33/573G01N33/53
CPCC07K7/06C12Q1/485C12Q1/48C07K7/08
Inventor GOUELI, SAID A.
Owner PROMEGA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap